The present invention relates to a triterpenoid cyclase (tetrahymanol cyclase) isolated from Tetrahymena, its coding nucleic acid, its production, and use.
The inventive triterpenoid cyclase catalyzes the formation of tetrahymanol from squalene by a direct cyclization of squalene (Capsi et al. (1968) J. AM. CHEM. SOC. 90:3563-3564; Abe et al. (1993) CHEM. REV. 93:2189-2206). The triterpenoid cyclase also recognizes oxidosqualene as a substrate (Abe and Rohmer (1994) J. CHEM. SOC. PERKIN TRANS. 1:783-791). In addition to pentacyclic triterpenoids, the squalene tetrahymanol cyclase also catalyzes the formation of tetracyclic triterpenoids (Abe and Rohmer (1991) J. CHEM. SOC. CHEM. COMMUN. 902-903). Tetrahymanol (or gammaceran-3-ol), which is derived from isoprene, is a member of the isoprenoid class. Isoprenoids play an important role as phytohormones and carotenoids, and as components of chlorophyll, ubiquinone, plant resins, oils, and latex. As steroid hormones, isoprenoids effect important functions in animals. The formation of tetrahymanol can be reprimed in Tetrahymena by adding sterols, such as cholesterol (Conner et al. (1968) J. PROTOZOOL. 15:600-605; Conner et al. (1969) J. BIOL. CHEM. 244:2325-2333).
Isoprenoids are also important components of bacterial and eukaryotic membranes. Similar to hopanoids and sterols (such as cholesterol), pentacyclic triterpenoid has tetrahymanol membrane-stabilizing properties (Conner et al. (1968; 1969); Poralla et al. (1980) FEBS LETT. 113:107-110). By restricting the fluidity of the lipid acid residues of membrane lipids, a condensed (membrane-solidifying) effect is achieved above the phase transition temperature; while below the phase transition temperature, the fluidity of the membrane is increased, thus preventing the optimal close packing of fatty acid residues. In addition, the membrane fluidity depends on the fatty acid composition of the membrane lipids. The fluidity of membranes increases in proportion to the levels of unsaturated fatty acids. With temperature changes, organisms are able to regulate the fluidity of their membranes, for example, via the fatty acid composition. Below the phase transition temperature, isoprenoids and unsaturated fatty acids increase the membrane fluidity via a synergistic effect. The inhibition of the synthesis of the cyclic triterpenoids alters membrane stability. This reduced membrane fluidity can be compensated by an increased proportion of polyunsaturated fatty acids (PUFAs) in the membrane, i.e., the content of PUFAs can be increased by inhibition of the triterpenoid cyclase.
The targeted modification of the composition of the fatty acid spectrum by means of gene technology for the commercial production of special fatty acids or oils is described in Napier et al. (CURR. OPIN. PLANT BIOL. (1999) 2:123-127); Murphy and Piffanelli (Soc. EXP. BIOL. SEMIN. (1998) Ser. 57 (PLANT LIPID BIOSYNTHESIS) 95-130); and FACCIOTTI and KNAUF (In: ADV. PHOTOSYNTH. 6: LIPIDS IN PHOTOSYNTHESIS: STRUCTURE, FUNCTION AND GENETICS. Siegenthaler and Murata (eds.) Kluwer Academic Publishers, Netherlands. (1998) 225-248). Thus, the modification of fatty acid composition can be regulated by altering the genes that code for enzymes which directly participate in the fatty acid synthesis, such as desaturases. However, it has been reported that the level of PUFAs in transgenetic organisms was relatively low (Knutzon and Knauf (1998) SOC. EXP. BIOL. SEMIN. SER. 67:287-304).
The knockout or repriming of the gene that codes for triterpenoid cyclase and the resulting deficiency of tetrahymanol may influence membrane fatty acid composition. However, the modified membrane fluidity can be balanced by the production of unsaturated fatty acids.
Although the triterpenoid cyclase protein from Tetrahymena is known and has been purified (Saar et al. (1991) BIOCHEM. BIOPHYS. ACTA, 1075:93-101), it had not been possible to clone the gene for triterpenoid cyclase from Tetrahymena (dissertation of Michal Perzl (1996) at the Faculty of Biology of Eberhard Karls University Txc3xcibingen). In previous studies, the gene sequence of triterpenoid cyclase could not be determined by sequencing the purified protein, PCR with degenerative primers, or hybridization with heterologous probes.
The present invention relates to nucleic acids isolated from Tetrahymena which code for a ciliate-specific triterpenoid cyclase. The inventive nucleotide sequences and the polypeptide sequences derived therefrom demonstrate a surprisingly minimal sequence identity to known isoprenoid cyclases. The invention also relates to the use of nucleic acids for the regulation of triterpenoid cyclase expression in a host organism, as well as the targeted knockout or repriming of the triterpenoid cyclase gene. As a result of the altered expression of the triterpenoid cyclase, it is possible to modify and enrich the levels of multiple unsaturated fatty acids in the host organism.
The present invention is directed to an isolated nucleic acid comprising a nucleic acid sequence encoding a polypeptide or functional variant thereof comprising the amino acid sequence of SEQ ID No. 12.
In a preferred embodiment, the isolated nucleic acid of the present invention comprises the nucleic acid sequences of SEQ ID No. 11 and SEQ ID No. 13. In another embodiment of the present invention, the isolated nucleic acid comprises at least 8 nucleotides of SEQ ID No. 11. Another embodiment is an isolated nucleic acid of the present invention wherein the nucleic acid is selected from the group comprising DNA, RNA, and double-stranded DNA. In yet another embodiment of the invention, the isolated nucleic acid comprises one or more non-coding sequences.
In one aspect of the invention, the isolated nucleic acid is antisense. Another aspect relates to a vector comprising the isolated nucleic acid of the present invention, preferably the vector is an expression vector. In addition, the invention is also directed to isolated host cells comprising said vector. Preferably, the host cells are protozoan, in particular, ciliate.
Another embodiment is a method of producing the isolated nucleic acid of the present invention comprising the step of chemically synthesizing said nucleic acid. An additional embodiment is a method of producing the isolated nucleic acid comprising the step of isolating said nucleic acid from a gene library by screening said library with a probe.
The present invention also relates to an isolated polypeptide or functional variant thereof comprising the amino acid sequence of SEQ ID No. 12. In particular, the invention relates to an isolated polypeptide comprising at least 6 amino acids of SEQ ID No. 12.
Also within the scope of the present invention is a method of producing a polypeptide comprising culturing a host cell under conditions sufficient for the production of said polypeptide and recovering said polypeptide from the culture. The host cell may be a protozoa, preferably a ciliate.
One aspect of the present invention is directed to an antibody capable of binding the polypeptide of SEQ ID No. 12. Another aspect of the present invention relates to a method of producing said antibody of comprising the steps of immunizing a mammal with a polypeptide and isolating said antibodies.
In one embodiment of the present invention, the isolated nucleic acid is used to identify polypeptide variants comprising the steps of screening a gene library with said nucleic acid and isolating said variant.
Also within the scope of the present invention is a method of enriching the saturated fatty acid content, in particular the squalene content, in a host cell comprising the step of inactivating the inventive nucleic acid. The nucleic acid may be inactivated by an antisense nucleic acid, by a deletion or insertion of a nucleic acid sequence, or mutation of said nucleic acid sequence. In particular, the inventive nucleic acid may be replaced with one or more selectable markers.
In another embodiment of the present invention, the isolated nucleic acid is used to produce cyclic triterpenoids, preferably pentacyclic triterpenoids, and most preferred tetrahymanol.